Project Summary Our goal is to develop a novel, immunologically enhanced L. lactis probiotic-based therapeutic for the treatment of Sjgren?s Syndrome (SjS). SjS is a progressive, chronic autoimmune disease characterized by inflammatory cell infiltration of the salivary and lacrimal glands, resulting in acinar epithelial cell atrophy, cell death, and loss of exocrine function1-6. At least half of SjS patients develop extraglandular inflammatory disease and have a wide range of systemic clinical manifestations that can affect any organ system, including connective tissue, and 5-10% of patients develop life-threatening lymphoma7, 8. SjS is a debilitating disease affecting as many as 3.1 million individuals in the U.S.9, 10, with women being nine times more likely to be afflicted with SjS than men5, 10, 11. Treatment of SjS remains a significant unmet medical need. Current treatment relies on replacement therapies such as artificial saliva and eye lubricants or immunosuppressive agents12, 13. Because of the multiple antigens involved in this disease process, i.e., ?-fodrin14-17, ribonuclear protein Ro/SSA14, 48-50, La/SSB14, 48, 49, and M3R18, 49, 51, oral tolerance methods become problematic. Thus, the capacity to stimulate regulatory cells independent of knowing the antigen specificity for the disease poses as an attractive therapeutic approach. Originally conceived as a human diarrheal vaccine, we found that colonization factor antigen I (CFA/I) from human enterotoxigenic E. coli (ETEC) is effective at inducing auto-Ag-specific T regulatory cells when administered orally as either a purified protein or delivered via a Salmonella or L. lactis bacterial delivery system19-21, 63. To avoid challenges associated with producing large quantities of CFA/I protein and improve the mucosal pharmacokinetic (PK) and pharmacodynamic (PD) properties of CFA/I following oral administration, we developed and characterized a vector-containing L. lactis-CFA/I expressing product (referred to as VTC- CFA) and evaluated its efficacy in multiple autoimmune models. Indeed, VTC-CFA was effective at reducing clinical symptoms in an SjS animal model, along with experimental models of RA and MS22-24. In the SBIR Phase I, we determined the gastrointestinal (GI) PK and optimal effective dose of VTC-CFA in a murine model of SjS. In addition, we developed our clinical candidate that expresses CFA/I from a genome-integrated operon (referred to as VTC-CFA-001) and completed an FDA pre-IND meeting to finalize our IND-enabling plans. This application is designed to advance VTC-CFA towards the clinic. The key aims of this proposal are: 1) optimize VTC-CFA-001 upstream process development for consistent manufacturing, 2) prepare GMP master and working cell banks to support VTC-CFA-001 production, 3) manufacture VTC-CFA-001 to perform dose optimization and toxicology, 4) Determine optimal dose of VTC-CFA-001 and identify/characterize biomarkers, and 5) complete VTC-CFA-001 GLP toxicology studies to enable IND filing. Successful commercialization of VTC-CFA-001 will provide a profound medical advancement for treating SjS.